In the majority of the adult skeleton, bone formation is confined to specific remodeling sites. Whether bone is lost or gained will depend on the extent of bone formation in each one of these sites which is thought to be regulated by local growth factors. Of the several growth factors present in bone, there is now sufficient evidence to document an important role for insulin-like growth factors (IGFs) in the local regulation of bone formation. Our past studies on regulation of IGF actions in bone have led to the purification and characterization of two new IGF binding proteins; IGFBP-4 and IGFBP-5. The primary goal of this project is to extend our past work and focus on the regulation and action of IGFBP-4 and IGFBP-5 and their proteases since IGFBP-4 is the major IGFBP produced by human bone cells and inhibits IGF induced bone cell proliferation and IGFBP-5 is the major IGFBP stored in bone and stimulates IGF induced bone cell proliferation. In this regard, we will test 3 relevant hypothesis: 1) That systemic and local effectors of bone metabolism could modulate IGF action by regulating the concentration of IGFBP-4 and IGFBP-5 at one or more regulatory sites in the metabolic sequence of IGFBP synthesis, secretion and degradation. To determine the cause and effect relationship between IGFBP production and modulation of bone cell proliferation, we will use an antisense oligodeoxyribonucleotide approach to evaluate the role of IGFBP-4 and IGFBP-5 in mediating the effects of selected systemic hormones. 2) That the concentration of IGFBP-4 and IGFBP-5 in the bone cell microenvironment is regulated in part by production of IGFBP specific proteases. In this regard, we propose studies to purify and characterize the recently discovered putative IGFBP-4 and IGFBP-5 proteases from human bone cell conditioned medium and determine the role of these proteases in regulating the concentration of IGFBP-4 and IGFBP-5. 3) That IGFBP-4 and IGFBP-5 modulate the biological effects of IGFs in bone cells either by altering the affinity of IGFs for IGF receptors or by acting through a IGFBP specific cell surface receptor. For these studies, we will use partially purified IGF receptors and IGF mutants that bind to IGF receptors but not to IGFBP-4 or IGFBP-5 and vice versa. All of the proposed work will involve human IGFBPs and IGFBP proteases using human osteoblast like cells. It is anticipated that these studies will provide valuable information regarding the regulation and actions of IGFBP-4 and IGFBP-5 in bone and should lead to major insights regarding the pattern of responses that are seen by different agents within the IGF system.